Fig. 3

Representation of in vitro schematic potential catalytic LPMO pathways for polysaccharide substrate hydroxylation. Pathways are designated with Roman numerals (i–vii). Pathway (i): reducing agents, substances that provide electrons and are subsequently oxidized, may produce non-enzymatically H₂O₂ over time, which may be intensified in certain conditions, such as high pH environment. Pathway (ii): in this pathway, reducing agents reduce LPMO’s active site, transitioning the copper at the active site of the enzyme from its oxidized state of LPMO, Cu(II) is reduced to Cu(I) by redox partners, leading to the formation of the intermediate LPMO–Cu(II)-O₂^–. This species further transforms into LPMO–Cu(II)-OOH^–, which, through interaction with another ascorbate molecule, resulting in the generation of H₂O₂ and Cu(I). Eventually, the Cu(I)- H₂O₂ intermediate converts to LPMO–Cu(II)-O^• –, which is the final intermediate depicted in scheme (All the intermediates are indicated by an asterisk in the scheme). Pathway (iii): this pathway demonstrates the peroxygenase H₂O₂-driven activity of LPMOs for the hydroxylation of polysaccharide substrates (e.g., cellulose). Pathway (iv): τhe monooxygenase activity of LPMOs is driven by molecular O₂. Pathway (v): LPMO-catalyzed reactions can undergo enzyme inactivation, likely due to auto-oxidation, a process where the LPMO becomes oxidized and thus loses its activity. Reduced LPMOs, whether the reaction is driven by O₂ or H₂O₂, are vulnerable to oxidative self-inactivation, especially in the presence of excess H₂O₂ concentrations. Pathway (vi): catalase effectively decomposes H₂O₂ into water and O2, significantly mitigating the risk of oxidative damage. This action minimizes or eliminates H₂O₂—dependent pathways, such as Pathway (iii), altering the possible reactions that LPMOs can partake in. Catalase interfered reactions are presented with brown colored shapes